PR-immunoprecipitated DNA from 5min R5020-treated cells
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were incubated in 1% Formaldehyde for 10min at 37ºC. After quenching with glycine, cells were whased once in ice cold PBS and collected in PBS plus protease and phosphatase inhibitors using a scrapper. Cells were lysed in two secuential steps to release nuclear contet and inmediately sonicated in Bioruptor. Sonication was evaluated by agarose gel electrophoresis, considering it succesful when fragments were homogenously distrubuted between 200-400bp. Enrichment of receptor-bound DNA was achieved by incubation with specific antibodies against either PR or ERalfa and immunoprecipitation with protein A beads. Libraries were prepared by the Genomics unit of the CRG Core Facility (Centre for Genomic Regulation, Barcelona, Spain) with NEBNext® ChIP-Seq Library Prep Reagent Set (ref. E6200S Illumina). Libraries were quality checked and quantified for single-end sequencing.